Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex expressed primarily in the liver, kidney, and intestine. The complex is consisted of a glucose-6-phosphate transporter (G6PT) that translocates G6P from cytoplasm to the lumen of the endoplasmic reticulum (ER) and G6Pase-alpha that hydrolyses G6P to glucose. Together, they maintain interprandial glucose homeostasis. Deficiencies in G6Pase-alpha and G6PT cause GSD-Ia and GSD-Ib, respectively. Both manifest the symptoms of G6Pase-alpha deficiency characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-Ib patients also present with neutropenia and myeloid dysfunctions. The current dietary therapy can not treat the underlying disease and long-term complications develop in adult patients. An understanding of the molecular genetics and pathogenesis of GSD-I is needed to lead to therapies that can rectify the long-term complications of GSD-I. Currently, only liver, kidney and intestine are considered to be involved in interprandial glucose homeostasis because the absence of G6Pase-alpha outside of these organs. We identified a ubiquitously expressed G6P hydrolase, G6Pase-beta that couples functionally with G6PT to form an active G6Pase complex. Our findings challenge the current dogma that only liver, kidney and intestine can contribute to blood glucose homeostasis. In addition to disrupted glucose homeostasis, GSD-Ib patients manifest intermittent neutropenia and myeloid dysfunctions. We generated a G6PT knockout (G6PT-/-) mouse that mimics all known defects of the human disorder and used the model to further our understanding of the pathogenesis of GSD-Ib. We demonstrate that the neutropenia is caused directly by the loss of G6PT activity; that chemotaxis and calcium flux, induced by the chemokines KC and macrophage inflammatory protein-2, are defective in G6PT-/- neutrophils; and that local production of these chemokines and the resultant neutrophil trafficking in vivo are depressed in G6PT-/- ascites during an inflammatory response. The bone and spleen of G6PT-/- mice are developmentally delayed and accompanied by marked hypocellularity of the bone marrow, elevation of myeloid progenitor cell frequencies in both organs and a corresponding dramatic increase in granulocyte colony stimulating factor levels in both GSD-Ib mice and humans. So, in addition to transient neutropenia, a sustained defect in neutrophil trafficking due to both the resistance of neutrophils to chemotactic factors, and reduced local production of neutrophil-specific chemokines at sites of inflammation, may underlie the myeloid deficiency in GSD-Ib. G6Pase-alpha is a highly hydrophobic, multiple domain transmembrane ER proteins, which can not be expressed in soluble forms. Therefore, protein replacement therapy for GSD-Ia is not an option, although somatic gene therapy, targeting G6Pase-alpha to the liver and the kidney, is an attractive possibility that has received interest recently. Using a G6Pase-alpha knockout (G6Pase-alpha-/-) mouse model generated in this laboratory, we have shown that a combined adeno (Ad-mG6Pase-alpha) and adeno-associated virus (AAV) serotype 2 (AAV2-mG6Pase-alpha)-mediated gene transfer leads to sustained G6Pase-alpha expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. Because AAV2-mG6Pase-alpha-mediated transgene expression increases slowly, typically peaking 5 to 7 weeks post-infusion, we co-infused AAV2-mG6Pase-alpha with Ad-mG6Pase-alpha which allows the infused animals to survive weaning. We now demonstrate that neonatal G6Pase-alpha-/- mice infused only with AAV1-mG6Pase-alpha, a recombinant AAV of serotype 1, not only survived weaning but also lived to 12 months of age. The AAV1-mG6Pase-alpha-mediated gene transfer leads to sustained G6Pase-alpha expression in both the liver and the kidney and corrected the murine GSD-Ia disorder for the full 12 months of the study. Our results suggest that human GSD-Ia would be treatable by gene therapy. Between meals, the primary source of blood glucose is gluconeogenesis and glycogenolysis. In the final step of both pathways G6P is hydrolyzed to glucose by the G6Pase-alpha complex. Since G6Pase-alpha is primarily expressed only in the liver, kidney and intestine, it has implied that most other tissues cannot contribute to interprandial blood glucose homeostasis. We demonstrate that a novel, widely expressed, G6Pase-related protein, PAP2.8/UGRP, renamed G6Pase-beta, is an acid labile, vanadate-sensitive, ER-associated phosphohydrolase, like G6Pase-alpha. Both enzymes have the same active site structure and exhibit similar kinetic properties. Most importantly, G6Pase-beta couples with the G6P transporter to form an active G6Pase complex that can hydrolyze G6P to glucose. Our findings challenge the current dogma that only liver, kidney and intestine can contribute to blood glucose homeostasis and explain why GSD-Ia patients, who lack a functional liver/kidney/intestine G6Pase-alpha complex, are still capable of endogenous glucose production. It has been shown that during G6P hydrolysis, G6Pase-alpha, a 9-transmembrane domain protein, forms a covalently bound phosphoryl-enzyme intermediate through His176, which lies on the lumenal side of the ER membrane. We now show that G6Pase-beta is also a 9-transmembrane domain protein that forms a covalently bound phosphoryl-enzyme intermediate during G6P hydrolysis. Using [32P]-G6P labeling, coupled with cyanogen bromide mapping, we demonstrate that the phosphate acceptor in G6Pase-beta is His167 and that it lies inside the ER lumen with the active site residues, Arg79 and His114. Therefore G6Pase-alpha and G6Pase-beta share a similar active site structure, topology, and mechanism of action. The demonstration of a significant, specific G6P hydrolase that can couple to G6PT outside of the liver raises interesting questions about the ability of other tissues to cycle glucose and contribute to blood glucose homeostasis. Of particular interest is the muscle, which expresses an elevated level of G6Pase-beta and stores the majority of body glycogen. We now demonstrate that muscle expresses both G6Pase-beta and G6PT and that they can couple to form an active G6Pase complex. Our data suggest that muscle may have a previously unrecognized role in interprandial glucose homeostasis. The islet-specific G6Pase-related protein (IGRP) is a member of the G6Pase family that is expressed primarily in the pancreatic islets and has low or non-detectable hydrolase activity. Recently, IGRP amino acids 206 to 214 was identified as a source of the nona-peptide recognized by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice, a model of type 1 diabetes. We show that IGRP is a glycoprotein, held in the endoplasmic reticulum by 9-transmembrane domains, which is degraded in cells predominantly through the proteasome pathway that generates the major histocompatibility complex class I-presented peptides. The divergence between amino acids 206 to 214 in IGRP and the corresponding sequence in mammalian G6Pase-alpha may explain why G6Pase-alpha, processed by the same pathway, is not presented to the immune system in an antigenic manner.